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Abstract. – OBJECTIVE: The warm isch-emia-reperfusion injury confines the prevalence of allografts. To improve the success rate of al-lotransplantation, we designed experiments to study the mechanism of calcium-calmodulin-de-pendent protein kinase type 2 (CaMK II) in isch-emia-reperfusion (I/R) injury.

MATERIALS AND METHODS: We established the I/R model in SD rats and performed the liver transplantation (LT). As a result, the expression of CaMK II in tissues was detected. CaMK II was interfered with and overexpressed by the transference of the lentivirus vector, and the he-patocyte apoptosis and viability were inspected. At the same time, the content of cytochrome c and apoptosis-inducing factor (AIF) were de-termined. The measurement of mitochondrial membrane potential and detection of intercellu-lar calcium levels were performed.

RESULTS: The expression of CaMK II signifi-cantly increased and is highly corresponded with the duration of warm ischemia. In BRL-3A cells and liver tissues, increased cellular apoptosis and less viability had been observed in the CaMK II overexpression group. Cytochrome c and AIF were also largely increased compared to the interfered group. Moreover, apparent mitochon-drial membrane potential loss has also been detected in the CaMK II overexpression group.

CONCLUSIONS: It suggested that CaMK II in-duces cell apoptosis. Our findings may give a nov-el indication that inhibition of CaMK II could be a new way for the therapy of warm ischemia-reper-fusion injury after LT in future clinical practice.

Key Words:Calcium-calmodulin-dependent protein kinase

type 2, Ischemia-reperfusion injury, Liver transplan-tation, Apoptosis.

Abbreviations

I/R injury, ischemia reperfusion injury; LT, liver trans-plantation; CaMK II, calcium-calmodulin-dependent protein kinase type 2; AIF, apoptosis inducing factor;

TUNEL, TdT-mediated dUTP nick end labeling; WIT, warm ischemia time; DCD, donation after circulatory death; Min, minute(s); H, hour(s); IHC, immunohis-tochemical; WB, Western blot; qRT-PCR, quantita-tive reverse transcription polymerase chains reaction; TUNEL, terminal-deoxynucleoitidyl transferase medi-ated nick end labeling; H&E, hematoxylin-eosin; rpm, evolution per minute; FCCP, carbonyl cyanide 4-(tri-fluoromethoxy) phenylhydrazone; LV, lentivirus; NC, negative control; Sh, short hairpin RNA; ALT, alanine aminotransferase; AST, aspertate aminotransferase; SD rat sprague dawley rats; PBS, phosphate-buffered sa-line; ER, endoplasmic reticulum; ROS, reactive oxygen species; RIPA, radioimmunoprecipitation assay; TBST, Tris-Buffered Saline and Tween-20; DMEM, Dulbecco’s Modified Eagle’s Medium; PI, propidium iodide; BSA, bovine serum albumin; D, day(s); GEO, gene expression omnibus; PVDF, polyvinylidene difluoride; DMSO, di-methyl sulfoxide.

Introduction

Worldwide, donor liver donations after cir-culatory death (DCD) has been endorsed for de-cades, and it has become an increasing source of allografts1-4. As reported, liver from DCD occu-pied about 30% of all liver transplants5. However, the use of DCD liver for transplantation in clini-cals remains cautious due to the WIT, which is the most important risk factor affecting tissue viabil-ity and graft function6,7. The DCD donors often suffer from trauma, hemorrhagic shock or cardi-ac arrest, this low-flow perfusion status is known as warm ischemia8. During the warm ischemia, livers from DCD donors inevitably go through hypotension, hypoxia, and ischemia/reperfusion injury9,10. Under this condition, liver after trans-plant may undergo varying degrees of damages with the prolonged warm ischemia, and the liver is more liable to suffer from primary graft dys-function and biliary complications11-13. Therefore, a method to recover the function of the liver that

European Review for Medical and Pharmacological Sciences 2020; 24: 3331-3343

W. LI, X.-B. ZHANG, R.-C. WU, S.-N. ZHANG, J. LIU, Y. GAO, K.-P. ZHENG, J.-H. RAN

Department of Hepatobiliary Pancreas and Vascular, Ganmei Hospital Affiliated to Kunming Medical University, Kunming, Yunnan, China

Corresponding Author: Jiang-Hua Ran, MD; e-mail: [email protected]

Calcium-calmodulin-dependent protein kinase type 2 induces apoptosis of hepatocytes after liver transplantation

W. Li, X.-B. Zhang, R.-C. Wu, S.-N. Zhang, J. Liu, Y. Gao, K.-P. Zheng, J.-H. Ran

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suffers from warm ischemia reperfusion injury is the foundation to which DCD can be used in clin-ical trials.

Current research has proven that I/R injury is able to cause transition in mitochondrial mem-brane potential (∆ψm)14,15. Furthermore, when the mitochondrial permeability transition pore opens, Ca2+ overloading leads to mitochondria swelling, and mitochondrial integrity is impaired and proapoptotic factors are release into the cyto-plasm16,17. Ultimately, these pathological changes accelerated cell death. Therefore, mitochondrial dysfunction is closed related to the I/R injury, and the target to timely maintain the mitochon-dria stability can protect the tissue survival from post-transplantation8.

CaMK II is a serine/threonine protein ki-nase and it is capable of acquiring the increasing intracellular Ca2+. When combined with Ca2+/CaM-binding, CaMK II undergoes activation by autophosphorylation; activating CaMK II can phosphorylate certain enzymes and regulates cell activity18,19. The activation of CaMK II has a hand in the regulation of cell apoptosis20-22. Join-er et al23, it also has been verified that the expres-sion of CaMK II promotes the apoptosis of myo-cardial cell after suffering from warm ischemia reperfusion injury. However, the mechanism of CaMK II regulation of post-liver transplantation is not clear.

In this paper, we reported the role of CaMK II in the liver, which was used in transplantation. Our data revealed overexpression of CaMK II could induce hepatocytes apoptosis. It will pro-vide a candidate target for the treatment of isch-emia-reperfusion injury in future clinic trials.

Materials and Methods

Animal CareSprague Dawley (SD) rats (Seven-week old,

250-300 g) were bought from Experimental Ani-mal Center of Kunming Medical University. They were placed in a pathogen-free room. The animal study protocol is legally approved by the Animal Care & Welfare Committee of Kunming Medical University. The approval number is: 2019-04. Ex-periments complied with the Guide that the Care and Use of Laboratory Animal published by the US National Institutes Health and the provisions of the Declaration of Helsinki. Experiments were performed under anesthesia that minimizes the pain of the animals.

Establishment of I/R Injury Model and Tissue Harvest

To determine if the expression of CaMK II is related to the WIT, we constructed the I/R inju-ry model of SD rats. The rats were anesthetized with isoflurane using cone masks (2% during the operation, 1 l/ml airflow, a fraction of inspiration O2 70%). A median and transverse laparotomy was performed in the supine position to expose the heart. 10 μl of heparin was dissolve in 1 ml of normal saline and injected into the rats through the dorsal vein of the penis. Finally, the thoracic aorta and superior vena cava were occluded with a traumatic vascular clamp to mimic the process of heart arrest for 0, 10 and 20 minutes (min). After the duration, 20 ml of cold Ringer’s lactate (Tuopai pharmaceutical Co., Ltd. Sichuan, China) was gently flushed into the livers. The livers were then isolated from the rats. The collected organs were stored in −80°C for further use. The trans-plantation operation was performed as previously described24.

Immunohistochemical (IHC) Staining

To detect the expression of CaMK II protein in liver tissue, the liver tissue samples which had been sliced and dewaxed were used for im-munohistochemical analysis. The sections were heated to repair the antigen, and hatched at room temperature with the peroxidase blocking re-agent for 10 min followed by washing with PBS (Hyclone, South Logan, UT, USA) for 3 × 3 min. The sections were then hatched with CaMKII antibody (Merck Millipore, Darmstadt, Germa-ny) for 12 h at 4°C and rinsed with PBS (Hy-clone, South Logan, UT, USA) for 3 × 3 min. Afterwards, the sections were cultivated with the secondary antibody (Proteintech, Rosemont, IL, USA) at normal temperature for 30 min. Af-ter repeating the process of washing with PBS (Hyclone, South Logan, UT, USA) 3 times, the sections were hatched using 3, 3’-diaminobenzi-dine (MaixinBiotechnique, Fuzhou, China) for 5 min. These sections were then washed with tap water and double steamed water for 10 min and 5 min respectively, and counterstained with hema-toxylin for 20 seconds. The sections were rinsed with hydrochloric acid alcohol for 5 min and flushed to return to blue. It was then dehydrated using gradient alcohol, cleared with xylene and mounted with neutral gum. In 24 hours (h), the sections were observed and photographed using an optical microscope.

CaMK II induces liver injury

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Western Blot (WB)To detect the level of CAMK II, AIF and cy-

tochrome c in BRL-3A cells and liver tissues, we preformed WB. Firstly, the BRL-3A cells and liv-er tissues were dealt with lysis/RIPA (Beyotime Biotechnology, Shanghai, China) buffer which containing 1% proteinase inhibitor for 5 min. Then, homogenates were centrifuged at 16000 rpm for 10 min at 4°C and purity of protein were measured by an assay kit (Bio-Rad, Hercules, CA, USA). After that, the acquisitions were isolated by gel electrophoresis and transferred to PVDF membranes, the membranes were blocked for 2 h in bovine serum albumin/TBST buffer (Becton Dickinson, Franklin Lakes, NJ, USA, 5% dried skimmed milk). Next, membranes were incubated with antibodies of CaMK II, AIF, and cytochrome c (Cell Signaling Technology, Inc., Danvers, MA, USA) for 12 h at 4°C, β-actin was adopted to an internal control. The membranes were washed thrice with TBST before reacting with secondary antibody (Protein Technology, Chicago, IL, USA) for 2 h at room temperature. Finally, the proteins were visualized scanned in LumiGlo substrate (Super Signal West Pico Trial Kit, Pierce, USA) with enhanced chemiluminescence (ECL).

Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR)

The expression of CAMK II mRNA was de-tected by the qRT-PCR method. As the first step, total RNA was extracted by TRIzol agentia (In-vitrogen Corp, Carlsbad, CA, USA), and its con-sistence and purity were determined. Then, the synthesis of cDNA is on the basis of reverse tran-scription kit (TaKaRa Biotechnology Co., Ltd., Komatsu, Japan). All primers for qRT-PCR were produced by Sangon (Sangon Biotech Co., Ltd., Shanghai, China), the sequences were showed in Table I. PCR was performed using a thermocycler: 95°C for 3 min, 95°C for 15 s, 60°C for 1 min, the total for 40 cycles. The data was analyzed by the comparative critical threshold (Ct) method, and the relative expression was analyzed by normal-ization to β-actin values using the 2-DDCt method.

GEO2R AnalysisFor further verification of the CaMK II ex-

pressed in the human liver tissues after LT, we screened the microarray data and corresponding clinical data of LT from the public Gene Expres-sion Omnibus (GEO) database: GEO accession number GSE14951. For this dataset, gene expres-sion in livers from DCD undergoing 50 ± 10 min of WIT was compared to the liver from the nor-mal patient. GEO2R (www.ncbi.nlm.nil.gov/geo/geo2r) was used to analyze the microarray data.

Cell Culture The rat hepatic cell lines BRL-3A were all

bought from the Chinese Academy of Sciences (Kunming, China) and melted in a water bath at 37ºC. After 2 min, the cell suspension was taken out and diluted at 1:4 (1 ml cell suspension, 4 mL medium) into the centrifuge tube at 1000 rpm for 3 min at room temperature. Then, cells were re-suspended by 2 ml medium. Next, the cells were sucked up and put into a culture flask containing 10 ml medium, the culture bottle was bred in the thermotank at 37°C and containing 5% CO2. Fi-nally, BRL-3A were raised in DMEM (Gibco, Grand Island, NY, USA).

CaMK II Overexpression and Downregulation

Lentivirus carrying scramble or LV-CaMK II or LV-sh-CaMK II construct was bought from Shanghai Genechem Co., Ltd. The following sequences were used: forward: 5’-GCGGTAG-GCGTGTACGGT-3’and reverse: 5’-ATTGTG-GATGAATACTGCC-3’ for LV-CaMK II; for-ward: 5’-TAATACGACTCACTATAGGG-3’ and reverse: 5’-CTGGAATAGCTCAGAGGC-3’ for LV-sh-CaMK II.

In vitro, BRL-3A cells were incubated in a 6-well plates and cultured in an incubator (5% CO2) at 37ºC. When the cell convergence rate was between 50% and 70%, lentivirus was added and cultured for 12 h. In vivo, lentivirus was transfect-ed into the liver by tail vein injection.

Annexin V/Propidium Iodide Staining Assay

Cells were incubated with 1×107 copies of LV-sh-CaMK II, LV-CaMK II, LV-sh-CaMK II-NC or LV-CaMK II-NC for 1 d. Cells apoptosis were identified byz Annexin V-FITC and propidium iodide (Becton Dickinson, Franklin Lakes, NJ, USA) dual staining. Briefly, cells were transferred to centrifuge tube and 1×105 cells were collected.

Table I. The primer sequences of qRT-PCR.

rat-CaMK II-S 5'-ACACAGGAATATGCTGCAAAAAT-3'rat-CaMK II-A 5'-GGAGCACAAACTGGAACAATGAC-3'rat-β-actin-S 5'-TGGGTATGGAATCCTGTGGCA-3'rat-β-actin-A 5'-GGCATTTCTGGAGATACGGTTGT-3'

S: sense; A: anti-sense.


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Then, cells were centrifuged at 2000 rmp for 5 min and re-suspended in 500 µL of 1× bind-ing buffer. Next, 5 µL of Annexin V-FITC was dropped in the cells and cells were hatched for 0.5 h at 4°C in the dark. Later, cells were incubated with 10 µL of propidium iodide for another 5 min. Apoptotic cells were then immediately measured by flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).

Cell Viability Assay BRL-3A cells were paved at a density of 5 ×

103 cells/well in 96-well plates for 24 h. After the adhesion, cells were transfected with 1×107 copies of LV-sh-CaMK II, LV-CaMK II, LV-sh-CaMK II-NC, and LV-CaMK II-NC. The cells were then cultured for another 1, 2, 3, 4, 5, and 6 days. Then, Cell Counting Kit-8 (CCK-8, Dojindo Molecular Technologies, Kumamoto, Japan) was drop in (10 μl/well), and the liquid was placed at 37°C for 30 min. Finally, the absorbance OD of the liquid was checked by a microplate reader at 450 nm.

Terminal-Deoxynucleoitidyl Transferase dUTPnick End Labeling (TUNEL) Staining

To detect apoptosis in liver tissue after LT, a one-step TUNEL Apoptosis Assay kit (Roche, Basel, Switzerland,) was used. Briefly, the 4 μm thick paraffin sections were dewaxed and air-dried. Then, proteinase K was mixed and treat-ed for 20 min. After that, it was incubated in a mixture of the fluorescent labeling solution of dUTP and the TdT enzyme at 37°C for 1 h. The sections in positive group were cultivated for 10 min with DNaseI at 25°C. The negative controls were bred with dUTP at room temperature for 10 min. The samples were then disposed with diam-inobenzidine (DAB), counterstained with hema-toxylin, dehydrated in a gradient series, vitrified with dimethyl benzene, and mounted with neutral balsam.

Hematoxylin-Eosin(H&E) StainingH&E staining was adopted to watch the patho-

logical changes of liver tissue after transplanta-tion. The method is as follows: after the rats were euthanized, the liver tissue sections were de-waxed and dehydrated, and hematoxylin solution was added and stained at room temperature for 10 min. The hematoxylin was washed away with wa-ter. 1 ml of 0.5% eosin solution was dripped into the sections, dyed at normal temperature for 60 min, and washed with running water for several seconds. The sections were then dehydrated with

graded alcohol and xylene. Neutral gum was used to seal the sections, and the pathological changes of liver tissue were observed by an optical micro-scope.

Liver Function TestAfter LT, the blood samples collected from the

orbit of rats. The contents of ALT and AST were analyzed by the automatic biochemical analyzer (Beckman AU5400, CA, USA).

Measurement of Mitochondrial Membrane Potential

Firstly, 1 ml of 1.0% protease XXIV in assay buffer was injected in fresh rat tissues. Tissues were homogenized and cells were harvested af-ter centrifuged at 1000 rpm for 10 min. Then, cells were transferred to HTF culture media that append 0.5% BSA (Hyclone, South Logan, UT, USA) after washed with 2 ml of PBS (Hyclone, South Logan, UT, USA) and incubated in > 85% humidity (37°C, 20% O2, 5% CO2). The cells of positive group were incubated with FCCP (Car-bonyl cyanide 4-(trifluoromethoxy) phenylhydra-zone) for 20 min before adding the JC-10, and the cells of treatment groups was hatched with acro-lein and CTX (25 μM) at incubator for 45 min. Then, the cells from all the groups were culti-vated with HOEST dye at 37°C for 20 min (20% O2, 5% CO2). For further analysis, each group of cells was washed, dissolved in 0.25% trypsin, and resuspended in PBS. Fluorescence signal was detected by flow cytometry on an LSR II-12 (Becton Dickinson, Franklin Lakes, NJ, USA) in green (emission: 525 nm) and red (emission: 590 nm) channels.

Detection of Intercellular Calcium LevelLiver tissues were treated as stated in mea-

surement of mitochondrial membrane potential. The lysis of Flou-3 AM probe (Sigma-Aldrich, St. Louis, MO, USA) was carried out in DMSO with a final concentration of 1 µM and added to the cell suspension. The sample was cultivated in incubator for 0.5 h. The sample obtained was well mixed and transferred into 10 equal micro-tubes, each tube contained 400 µl of the sample. To remove additional Flou-3 AM, all the micro-tubes were centrifuged for 5 min. The sediment was re-suspended to a volume of 500 ml using Ham’s F10 (Gibco, Grand Island, NY USA). All experimental groups were transferred to the flow cytometry tube. The data were calculated using the FlowJo Software.


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Statistical AnalysisThe data in the experiment were expressed as the

mean ± standard deviation of the three independent experiments and SPSS 19.0 was used for statistical analyses. Student’s t-tests were used to compare the averages of the two groups, and Bonferroni’s-cor-rected one-way ANOVA is used to compare the av-erages of three or more groups. *p<0.05 was indicat-ed as a statistical significance.

Results

CaMK II Expression in the Liver Tissues Is Significantly Increased After LT

To determine the expression change of CaMK II in liver tissues after transplantation, we first con-structed the I/R models with prolonged WIT. The WB, IHC staining, and qRT-PCR tests were also con-ducted. As a result, when the WIT was prolonged to 10 min and 20 min, CaMK II protein level was large-ly increased when compared with the normal group (Figure 1A and B, p<0.05). The CaMK II protein appeared strongly positive when the duration was 20 min as compared to the other two groups in D7 (Fig-ure 1C). Up-regulated CaMK II mRNA expression was observed in D7 (Figure 1D, p<0.05). GEO2R analysis showed that the CaMK II expression was higher in DCD patients than the norm patients (Fig-ure 1E, p<0.05), which was the same as our findings. The above observation indicated CaMK II expression level significantly increases after LT.

The Apoptosis of BRL-3A Cells Increased With the Increase of CaMK II, and Decreased With the Decrease of CaMK II

To analyze the effects of CaMK II on hepato-cyte, we applied lentivirus carrying sh-RNA specifically targeting CaMK II, and lentivirus carrier overexpression of CaMK II carriers to transfer BRL-3A cells. Mock-vehicle groups and saline groups were designed as control groups. Western blot confirmed that LV-sh-CaMK II and LV-CaMK II succeeded in significantly knocking down and overexpressing CaMK II in cell lines (Figure 2A and B). Flow cytometry assay show that the apoptosis frequency of BRL-3A cells were increased in the LV-CaMK II group and de-creased in LV-sh-CaMK II group compared with control group (Figure 2C, p<0.05). The S phase of BRL-3A cells were prolonged in LV-sh-CaMK II group and shortened in LV-CaMK II group when compared to the control group (Figure 2D). Simul-taneously, the CCK8 assay detected the cell via-

bility was increased after inhibiting the CaMK II (Figure 2E, p<0.05). CaMK II induction appears to reduce BRL-3A cells survival, and CaMK II inhibited increase BRL-3A cells proliferation.

Cytochrome c and AIF Increase With the Overexpression of CaMK II

To evaluate whether CaMK II augmented apoptosis through cytochrome c /AIF activa-tion, we overexpressed and inhibited CaMK II in BRL-3A cells and detected the expression of cy-tochrome c and AIF by WB. Indeed, the overex-pression of CaMK II enhanced the expression of cytochrome c and AIF. After interfering with the expression of CaMK II, the level of cytochrome c and AIF has declined accordingly (Figure 3A to D, p<0.05). It confirmed CaMK II can regulate the level of cytochrome c and AIF, and partici-pates in the progress of hepatocyte apoptosis.

The High Level of CaMK II Is Accompanied by Increased Hepatocyte Apoptosis and Liver Function Injury In Vivo

The effects of CaMK II on liver transplantation in vivo were assessed using the liver transplantation models of SD rats with 10 min of WIT. Lentivirus with a CaMK II over-expressing sequence (LV-CaMK II) or sh-CaMK II (LV-sh-CaMK II) was transfected into the liver by tail intravenous injec-tion. The rats’ serum for detecting the level of ALT and AST were collected from the 1st, 3rd and 7th day after the operation. The rats were euthanized at D7, and the data was analyzed. As a result, the fluores-cence microscope showed lentivirus with the carrier of LV-CaMK II and LV-sh-CaMK II was symmet-rically distributed in the liver (Figure 4A). qRT-PCR analysis confirms that CaMK II was interfered with and over-expressed successfully in the liver tissue (Figure 4B, p<0.01). TUNEL staining showed that the liver tissues appeared to have more cell apopto-sis in the LV-CaMK II group than the control group (p<0.05), and there is a lower apoptosis rate in LV-sh-CaMK II group (Figure 4C and D, p<0.01). The liver function test showed that the AST and ALT in serum were increased over time, and both AST and ALT were increased in the LV-CaMK II group at D7. On the contrary, the level of AST significantly declined at D7 in the LV-sh-CaMK II group com-pared to the control group (Figure 4E and F, p<0.01). The result of H&E staining indicates that there is an acidophilic change in 3 control groups, and spotty necrosis was found in the LV-CaMK II group. The LV-sh-CaMK II group represents the cell division phase (Figure 4G).


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These results illustrate that CaMK II is suc-cessfully overexpressed and suppressed in liver cells, and the up-regulated CaMK II induced the apoptosis of hepatocyte and liver function injury in SD rats. However, inhibition of CaMK II will induce the proliferation of hepatocytes and recov-ery of liver function.

The Up-Regulation of CaMK II Leads to Mitochondrial Dysfunction and Up-Regulation of Cytochrome c and AIF

For further study of the apoptotic pathway, we de-tected the level of cytochrome c and AIF, the change of mitochondrial morphology and membrane poten-tial, and the concentration of Ca2+ in rat hepatocytes.

Figure 1. The expression of CaMK II in the liver after I/R injury. A, The protein bands of CaMK II in D1, D3, and D7 after the I/R injury with different WIT (0 min, 10 min, and 20 min). CaMK II: calcium-calmodulin-dependent protein kinase type 2; D: day; WIT: warm ischemia time; min: minute(s); (n = 6/group). B, The histogram of CaMK II protein in D1, D3 and D7 after the I/R injury with different WIT (0 min, 10 min, and 20 min). CaMK II: calcium-calmodulin-dependent protein kinase type 2; D: day; WIT: warm ischemia time; min: minute(s); (n = 6/group, **p<0.01). C, The immunohistochemical of CaMK II in D1, D3, and D7 after the I/R injury with different WIT (0 min, 10 min, and 20 min). The brown dots represent marked CaMK II proteins. WIT: warm ischemia time; min: minute(s); (n = 6/group, scale bar = 100 μm). D, The relative expression of CaMK II mRNA in D1, D3, and D7 after the I/R injury with different WIT (0 min, 10 min, and 20 min). CaMK II: calci-um-calmodulin-dependent protein kinase type 2; D: day; WIT: warm ischemia time; min: minute(s); (n = 6/group, **p<0.01). E, The relative expression of CaMK II in normal liver cells and DCD liver cells. The green circles represent normal liver cells, and the purple boxes represent DCD liver cells. CaMK II: calcium-calmodulin-dependent protein kinase type 2; DCD: donation after circulatory death; (n = 8/group, scale bar = 50 μm, *p<0.05, **p<0.01).

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Figure 2. Effect of overexpression and inhibition of CaMK II on BRL-3A cells. A, The protein bands of CaMK II in 5 groups (Control, LV-CaMK II, LV-CaMK II-NC, LV-sh-CaMK II, and LV-sh-CaMK II-NC). CaMK II: calcium-calmodulin-dependent protein kinase type 2; LV: lentivirus; NC: negative control; sh: short hairpin RNA; (n=6/group). B, The WB column diagram of CaMK II protein in 5 groups. CaMK II: calcium-calmod-ulin-dependent protein kinase type 2; LV: lentivirus; NC: neg-ative control; sh: short hairpin RNA; (n = 6/group, **p<0.01). C, The apoptosis rate of BRL-3A cells in 5 groups. CaMK II: calcium-calmodulin-dependent protein kinase type 2; LV: lentivi-rus; NC: negative control; sh: short hairpin RNA; (n = 6/group, **p<0.01). D, The percentage of BRL-3A cells in S phase in 5 groups. CaMK II: calcium-calmodulin-dependent protein kinase type 2; LV: lentivirus; NC: negative control; sh: short hairpin RNA; (n = 6/group, **p<0.01). E, The viability of BRL-3A cells in 5 groups. CaMK II: calcium-calmodulin-dependent protein kinase type 2; LV: lentivirus; NC: negative control; sh: short hairpin RNA; (n = 6/group).


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Consistent with the cell experiment, WB analy-sis revealed after overexpressing CaMK II, the expression of cytochrome c and AIF was in-creased significantly. When interfering with the expression of CaMK II, the level of cytochrome c and AIF has decreased accordingly (Figure 4A to D, p<0.01). Transmission electron microscope observed that mitochondrial membrane integrity was destroyed, and the mitochondrial ridge was disordered in the LV- CaMK II group, compared to the control group (Figure 5E). Detection of mitochondrial membrane potential showed that

after the overexpression of CaMK II, the ratio of red/green cell was decreased compared with the control group (p<0.05). Contrastingly, the ratio of red/green cell appeared to ascend in the LV-sh CaMK II group significantly (Figure 5F, p<0.01). The measurement of the concentration of Ca2+ indicated that there was no discrepancy among the 4 groups (Figure 5G). Our results revealed that CaMK II activates cytochrome c/AIF pathway, and regulates the apoptosis of hepatocyte through promotion of mitochondrial dysfunction.

Figure 3. The expression of CaMK II, cytochrome c, and AIF in BRL-3A cells. A, The protein bands of CaMK II, cyto-chrome c, and AIF in 5 groups (Control, LV-CaMK II, LV-CaMK II-NC, LV-sh-CaMK II, and LV-sh-CaMK II-NC). CaMK II: calcium-calmodulin-dependent protein kinase type 2; AIF: apoptosis-inducing factor; LV: lentivirus; NC: negative control; sh: short hairpin RNA; (n = 6/group). B, The ratio of CaMK II/β-actin in 5 groups. CaMK II: calcium-calmodulin-dependent protein kinase type 2; LV: lentivirus; NC: negative control; sh: short hairpin RNA; (n = 6/group, **p<0.01). C, The ratio of cytochrome c/β-actin in 5 groups. CaMK II: calcium-calmodulin-dependent protein kinase type 2; LV: lentivirus; NC: negative control; sh: short hairpin RNA; (n = 6/group, **p<0.01). D, The ratio of AIF/β-actin in 5 groups. AIF: apoptosis-inducing factor; CaMK II: calcium-calmodulin-dependent protein kinase type 2; LV: lentivirus; NC: negative control; sh: short hairpin RNA; (n = 6/group, **p<0.01).

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Figure 4. Effect of overexpression and inhibition of CaMK II on apoptosis rate, pathological changes, and liver function. A, A fluorescence microscope observed the distribution of viral vectors in the liver. CaMK II: calcium-calmodulin-dependent protein kinase type 2; LV: lentivirus; sh: short hairpin RNA; (n = 6/group, Scale bar = 8 μm). B, Relative levels of CaMK II in 5 groups. CaMK II: calcium-calmodulin-dependent protein kinase type 2; LV: lentivirus; NC: negative control; sh: short hairpin RNA; (n = 6/group, **p<0.01). C, The TUNEL positive cells in the liver tissue in 5 groups. CaMK II: calcium-calm-odulin-dependent protein kinase type 2; LV: lentivirus; NC: negative control; sh: short hairpin RNA; (n = 6/group, scale bar = 50 μm). D, The percentage of TUNEL positive nuclei in the liver tissue in 5 groups. CaMK II: calcium-calmodulin-dependent protein kinase type 2; LV: lentivirus; NC: negative control; sh: short hairpin RNA; (n = 6/group, **p<0.01). E, The serum ALT on D3 and D7 in 5 groups. ALT: alanine aminotransferase; D: day; CaMK II: calcium-calmodulin-dependent protein kinase type 2; LV: lentivirus; NC: negative control; sh: short hairpin RNA. (n = 6/group, *p<0.05, **p<0.01). F, The serum AST on D3 and D7 in 5 groups. AST, aspartate aminotransferase; D: day; CaMK II: calcium-calmodulin-dependent protein kinase type 2; LV: lentivirus; NC: negative control; sh: short hairpin RNA; (n = 6/group, **p<0.01). G, Pathological observation of liver tissue in 5 groups. CaMK II: calcium-calmodulin-dependent protein kinase type 2; LV: lentivirus; NC: negative control; sh: short hairpin RNA; (n = 6/group, scale bar = 100 μm).

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Figure 5. The Study of the CaMK II mechanism regulating apoptosis of hepatocytes. A, The protein bands of CaMK II, cytochrome c, and AIF in 5 groups (Control, LV-CaMK II, LV-CaMK II-NC, LV-sh-CaMK II, and LV-sh-CaMK II-NC). CaMK II: calcium-calmodulin-dependent protein kinase type 2; AIF: apoptosis-inducing factor; LV: lentivirus; NC: negative control; sh: short hairpin RNA; (n = 6/group). B, The ratio of CaMK II/β-actin in 5 groups. CaMK II: calcium-calmodulin-dependent protein kinase type 2; LV: lentivirus; NC: negative control; sh: short hairpin RNA; (n = 6/group, **p<0.01). C, The ratio of cytochrome c/β-actin in 5 groups. CaMK II: calcium-calmodulin-dependent protein kinase type 2; LV: lentivirus; NC: negative control; sh: short hairpin RNA; (n = 6/group, **p<0.01). D, The ratio of AIF/β-actin in 5 groups. AIF: apoptosis-inducing factor; CaMK II: calcium-calmodulin-dependent protein kinase type 2; LV: lentivirus; NC: negative control; sh: short hairpin RNA; (n = 6/group, **p<0.01). E, Pathological changes of mitochondria in 5 groups. CaMK II: calcium-calmodulin-depen-dent protein kinase type 2; LV: lentivirus; NC: negative control; sh: short hairpin RNA; (n = 6/group, scale bar = 500 nm). F, The ratio of red: green of 5 groups. CaMK II: calcium-calmodulin-dependent protein kinase type 2; LV: lentivirus; NC: negative control; sh: short hairpin RNA; (n = 6/group, *p<0.05, **p<0.01). G, Measurement of the concentration of Ca2+ in intracellular in 5 groups. CaMK II: calcium-calmodulin-dependent protein kinase type 2; LV: lentivirus; NC: negative control; sh: short hairpin RNA; (n = 6/group).

A

C

E

F

D

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Discussion

In this study, we showed that CaMK II signifi-cantly increased after I/R injury, which cause mi-tochondrial dysfunction and pro-apoptotic factor release. Due to this, liver cells apoptosis occurs. The present research suggests that CaMK II might play a key role in I/R injury after LT, and cyto-chrome c, AIF might be its direct target factor, which provides a new direction for fundamental research and clinical therapeutic strategies.

We found the damage of liver tissues is closely related to the release of CaMK II. These findings reflected 3 aspects: (a) the expression of CaMK II is correlated with WIT. (b) CaMK II is persistently increasing after LT, and the peak can be postponed to D7. (c) The liver pathological changes are closely related to the overexpression of CaMK II. Existing studies have reported that the activation of CaMK II can be triggered on the condition of the ER stress, hyperglycemia, and oxidative stress25-27, and that CaMK II can increase in early time after I/R injury in cardiomyocytes14,28. This is the first investigation to link the expression of CaMK II with the I/R in-jury in DCD liver. Our findings indicated that the expression of CaMK II appeared to slowly grow, which may explain why the sustained damage of liver tissue is related to the expression of CaMK II.

Previous researches showed that Ca2+ can in-crease in the early stage of I/R injury29. The cause

of Ca2+ release is correlated with the ER stress20,30, the outcome of ER stress is aberrant Ca2+ re-lease31,32. Ca2+ participates in phospholipase, cal-pain activation and other functions33, it can regulate the activation of CaMK II. In turn, the phospholi-pase of CaMK II can regulate the influx of Ca2+ into the mitochondria18. Once the Ca2+ overloads in the mitochondria, the mitochondria respond to the change of mitochondrial permeability, and the proapoptotic factors were released34, then the he-patocyte initiates cell apoptosis.

CaMK II is a multifunction regulator protein, which plays a key role in the intercellular junction and the pathological process, including inducing the I/R injury in special tissue. There is enough evidence that CaMK II and mitochondrial apop-tosis can increase after I/R injury14,28. Joiner et al23 found that CaMK II activity can regulate the Ca2+

influx in mitochondria and accelerate cell apopto-sis. Li et al35 reported that CaMK II activity can lead to the impairment of astrocytes in ischemia neuron death. However, the mechanism of CaMK II functions as I/R injury in LT is still unknown. Our results over the I/R injury of DCD liver are listed on the model in Figure 6. The current study suggests that the expression of CaMK II promotes the apoptosis of hepatocyte21. Further research indicated that prolonged warm ischemia time in-creases the expression of CaMK II, which will lead to the overload of Ca2+ in the hepatocyte; a

Figure 6. Mechanism of CaMK II regulating hepatocyte apoptosis after I/R injury. I/R injury: ischemia reperfusion injury; LT: liver transplantation; CaMK II: calcium-calmodulin-dependent protein kinase type 2; AIF: apoptosis-inducing factor.


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report showed that the intracellular Ca2+ increas-ing is correlated with the ER stress when the cell undergoes ROS and damage. The intracellu-lar Ca2+ influx accumulation is regulated by the CaMK II20. Ca2+ overload will lead to mitochon-drial permeability transition, which will cause the mitochondrial permeability. Those changes result in the release of proapoptotic factors in the mitochondria, such as cytochrome c and AIF. It has been observed that cytochrome c and AIF can activate caspase 3, which is the important factor for cell apoptosis36,37. Finally, mitochondrial apop-tosis will be initiated, and cause the hepatocyte death. After inhibiting the expression of CaMK II, the mitochondrial apoptosis pathway will be sup-pressed. Therefore, the I/R injury will be alleviat-ed. Therefore, inhibiting the expression of CaMK II can be utilized in the I/R injury treatment after LT. Another finding shows that, after decreasing the expression of CaMK II, the hepatocytes ap-peared to proliferate, which was reflected in our in vitro and in vivo experiment. In vitro, hepato-cytes either appeared to reduce cell apoptosis or improve cell activity, and the cell cycle of the S phase started to extend. In vivo experiment, we can recognize the division stage of hepatocytes in the liver tissue. Our ongoing research managed to explain the proliferation phenomenon. Results re-garding hepatocarcinogenesis established that the deletion of CaMK II can enhance the activation of mTORC1 and promote the hyperproliferation of hepatocytes. However, the mechanism of CaMK II function as the hepatocytes suffering from I/R injury need further studies38.

Conclusions

We discovered that CaMK II is a promotor which accelerates the damage of I/R injury under-going prolonged WIT. The benefit of suppression of the expression of CaMK II in the DCD liver depends on: (a) decreasing the hepatocyte apopto-sis and alleviating the liver damage after LT; (b) promoting the hepatocyte proliferation and recov-ering from the warm ischemia injury.

AcknowledgementsThe authors thank Dr. Jiang-Hua Ran (Department of Hepatobiliary Pancreas and Vascular, Kunming Municipal First People's Hospital, Kunming, Yunnan, China) for the critical comment and discussion on this study. This research

was supported by the National Natural Science Foundation of China under the grant [Grant No. NSF 81660112]; and the Yunnan Provincial Science and Technology Depart-ment-Kunming Medical University under the grant [Grant No. 2015FA010].

Conflict of InterestsThe authors declare that they have no conflict of interests.

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